Vaccination is the most important method of preventing seasonal influenza virus infection and alleviating symptoms of disease. Currently, there are three types of licensed vaccines for human use: inactivated vaccines delivered intramuscularly (i.m.), live attenuated influenza vaccines (LAIVs) administered intranasally (i.n.), and recombinant hemagglutinin (HA) administered i.m. The licensed seasonal LAIV in the United States is a recombinant virus with the internal PB2, PB1, PA, NP, M, and NS gene segments (backbone) derived from a human influenza A/Ann Arbor/6/60 (H2N2) virus (AA) that is cold adapted (ca), temperature sensitive (ts), and attenuated (att) (2). The HA and NA genes in the vaccine virus are derived from an influenza virus that is antigenically matched to strains predicted to circulate during the upcoming influenza season. The temperature sensitive (ts) phenotype of the LAIV is characterized by restricted (>100-fold) replication at 39C. The ts genetic signature (ts signature) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts signature into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine influenza viruses. The VN04ts sig and CA07ts sig viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39C, but the replication of WSNts sig was restricted >100-fold compared to that at 33C. Reassortant CA07ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication >100-fold at 39C. In ferrets, the CA07ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses.